Transcriptional program of Kpna2/Importin-α2 regulates cellular differentiation-coupled circadian clock development in mammalian cells

Abstract

The circadian clock in mammalian cells is cell-autonomously generated during the cellular differentiation process, but the underlying mechanisms are not understood. Here we show that perturbation of the transcriptional program by constitutive expression of transcription factor c-Myc and DNA methyltransferase 1 (Dnmt1) ablation disrupts the differentiation-coupled emergence of the clock from mouse ESCs. Using these model ESCs, 484 genes are identified by global gene expression analysis as factors correlated with differentiation-coupled circadian clock development. Among them, we find the misregulation of Kpna2 (Importin-α2) during the differentiation of the c-Myc-overexpressed and Dnmt1 −/− ESCs, in which sustained cytoplasmic accumulation of PER proteins is observed. Moreover, constitutive expression of Kpna2 during the differentiation culture of ESCs significantly impairs clock development, and KPNA2 facilitates cytoplasmic localization of PER1/2. These results suggest that the programmed gene expression network regulates the differentiation-coupled circadian clock development in mammalian cells, at least in part via posttranscriptional regulation of clock proteins.T he circadian clock is an intrinsic time-keeping system that regulates essential physiological functions such as sleep/wake cycles, body temperature, and metabolism (1-3). In the mammalian clock system the central pacemaker resides in the suprachiasmatic nucleus of the hypothalamus, coordinating cell-autonomous molecular oscillators throughout the body to perform tissue-specific functions. The molecular oscillator consists of transcriptional/ translational feedback loops of clock genes. Two transcription factors, CLOCK and BMAL1, heterodimerize and transactivate core clock genes such as the Period (Per1, 2, 3), Cryptochrome (Cry1, 2), and Rev-Erbα genes via E-box regulatory elements. PER and CRY proteins, in turn, translocate into the nucleus to suppress CLOCK/BMAL1 activity, leading to cyclic expression of these clock genes (4-9). Furthermore, REV-ERBα negatively regulates Bmal1 transcription via the RORE promoter element, thus driving antiphasic expression patterns of Bmal1 (10, 11).Despite uncovering the emergence of the circadian rhythms that occur during development (12-14), the precise mechanism of circadian clock development in mammalian cells remains unclear. Recently it has been found that pluripotent ES cells (ESCs) do not display discernible circadian molecular oscillations, whereas in vitro-differentiated ESCs displayed robust circadian oscillations of reporter expression (15-17). Moreover, we also have shown that circadian oscillations were abolished when differentiated cells were reprogrammed to regain pluripotency by expression of reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc) (15). These results suggested that the emergence of the cell-autonomous circadian oscillator is coupled with cellular differentiation. Cellular differentiation, as well as reprogramming, results in global alterations of the transcriptional program and epi...

Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9‐based Rett syndrome model mouse

“…; Umemura et al . , ). We introduced a large deletion of protein coding sequences spanning across exons 3 and 4 in PER2 Luc knock‐in ESCs with the CRISPR/Cas9 system using two sgRNA‐targeting sites and obtained several mutant cell lines (Fig.…”

MYC Disrupts the Circadian Clock and Metabolism in Cancer Cells

“…During the course of our studies, a recent report documented a role for MYC in disrupting the emergence of the circadian clock during differentiation of embryonic stem cells (Umemura et al, 2014). However, in these normal cells, MYC disruption of the circadian clock was dominated by a block in PER nuclear import rather than a direct effect on circadian E-boxes as we demonstrated in cancer cell lines.…”

Cancer/Testis Antigen PASD1 Silences the Circadian Clock

“…It has yet to be demonstrated that testis from other mammals, including humans, do not have circadian rhythms; however, based on the data presented here, we speculate that high levels of PASD1 in human testis could lead to suppression of circadian rhythms in the germline (Cooper et al, 2006). Connections between the lack of circadian cycling in undifferentiated embryonic stem cells and the germline are just coming to light (Paulose et al, 2012; Umemura et al, 2014; Yagita et al, 2010), making PASD1 an interesting link that could be explored further.…”